Search Results (71)

Klebsiella oxytoca bacilli co-form the human intestinal microbiota, but in favorable conditions, they may also affect immunocompromised individuals, causing urinary tract infections, bacteremia, or antibiotic-associated hemorrhagic colitis. The growing numbers of clinical outbreaks of K. oxytoca infections make these bacteria an emerging pathogen, which is still masked by the predominant K. pneumoniae isolates. Thus, it is very important to advance knowledge on K. oxytoca pathogenicity. This work aims to characterize a urine isolate, K. oxytoca 0.062, from central Poland, which appears to present a multidrug-resistant and extended-spectrum β-lactamases-positive phenotype. The structural experiments include sugar and methylation analyses, mass spectrometry, and ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy. Additionally, ¹H,¹H ROESY, and ¹H,¹³C HMBC experiments were carried out on the high-molecular-weight O polysaccharide fraction of K. oxytoca lipopolysaccharides (LPSs). These analyses led to the detection of two polysaccharide antigens: one neutral, containing a linear trisaccharide unit called mannan, and one acidic, which is built up of a branched tetrasaccharide unit containing two mannopyranose (α-Manp) residues, one galactopyranose (β-Galp) residue, and one galacturonic acid (α-GalpA) residue. The GalpA residue seems to be a potential minor epitope, recognized by the selected Proteus antisera in the serological studies. Full article

Background: An emerging trend of mutual convergence between drug-resistant and highly virulent strains of K. pneumoniae has been identified, highlighting the urgent need for the development of novel vaccines. Methods: To delete the target genes and eliminate the plasmids carrying antibiotic resistance genes, CRISPR-Cas9 technology was employed to perform genome editing on a clinically isolated O2 serotype of K. pneumoniae. Subsequently, this strain was utilized as a host to express genes associated with the synthesis of O1 serotype LPSs to construct the recombinant strain capable of simultaneously expressing LPSs of both O1 and O2 serotypes. This recombinant strain was then used as the production strain for the preparation of vaccines based on GMMAs (Generalized Modules for Membrane Antigens), and its biological characteristics were characterized. Finally, the safety and immunogenicity of the vaccine were evaluated using mice as the model animals. Result: a GMMA vaccine characterized by a high yield and low toxicity was gained. Importantly, the lipopolysaccharides (LPSs) of both O1 and O2 serotypes of K. pneumoniae were successfully expressed on the surface of the outer membrane vesicles. Following immunization with the GMMA vaccine, mice were capable of producing antibodies against the GMMA and demonstrated resistance to the invasion of both serotypes of clinically isolated K. pneumoniae. Conclusions: The GMMA vaccine showed significant promise as a bivalent vaccine against K. pneumoniae. Full article

ΦV10 is an Escherichia coli O157:H7-specific bacteriophage that has been used to develop luminescent reporter assays for the detection of this important foodborne pathogen. Previous work demonstrated the specificity of ΦV10 for infection of E.coli O157:H7 through interaction with the O157 antigen. In addition, modification of the lipopolysaccharide (LPS) via O-acetylation prevents ΦV10 infection in an E. coli O157:H7 expressing a phage-encoded O-acetylase gene. Through assays for phage binding, plaque formation, and lysogeny using non-O157:H7 and O157: non-H7 strains, as well as complementation of an O157:H⁻ strain, it is demonstrated in this study that both the somatic O157 antigen and flagellar H7 antigen are required for productive infection of E. coli O157:H7 by ΦV10. Together, the results indicate that the O157 antigen is required for phage binding and that the H7 antigen is necessary to complete the infection process. Full article

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, can rapidly degrade crystalline cellulose through direct cell-to-substrate contact. Most of its cellulases are secreted by the Type IX secretion system (T9SS) and anchored to the cell surface. Our previous study proved that the C-terminal domain (CTD) of the T9SS substrate cellulase Cel9A is glycosylated in C. hutchinsonii. However, its glycosylation mechanism has remained elusive. In this study, we found that chu_3394, which encodes UDP-glucose 6-dehydrogenase (Ugd), was important for the glycosylation of large amounts of periplasmic and outer membrane proteins in C. hutchinsonii. The contents of mannose, glucose, galactose, and xylose were detected to be reduced in the glycoproteins of the ∆ugd mutant compared to that of wild-type. They might be essential monosaccharides that contribute to the structure and function of glycans attached to proteins in C. hutchinsonii. The depletion of mannose, glucose, galactose, and xylose indicates a decrease in glycosylation modifications in the ∆ugd mutant strain. Then, we found that the deletion of ugd resulted in weakened glycosylation modification of the recombinant green fluorescent protein-tagged CTD of Cel9A. Additionally, the outer-membrane localization of Cel9A was affected in the mutant. Besides this, Ugd was also important for the synthesis of O-antigen of lipopolysaccharide (LPS). Thus, Ugd was involved in the synthesis of glycans in both glycoproteins and LPS in C. hutchinsonii. Moreover, the deletion of ugd affected the cellulose degradation, cell motility, and stress resistance of C. hutchinsonii. Full article

Bacteria from the genus Proteus are facultative human pathogens, primarily attacking the urinary tract and wounds. A total of 85 O serogroups have been identified so far among these bacilli. P. mirabilis Bprz 86 was isolated from the fistula of a patient in Łódź, Poland. Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting studies involving the P. mirabilis Bprz 86 lipopolysaccharide (LPS) and the strain-specific rabbit antiserum indicated that the strain, which does not belong to any of the O1–O85 serogroups, shares a common epitope with Proteus O17 antigens and is identical to another clinical P. mirabilis strain, Sm 120, isolated from the urine of a patient in the area. The O-specific polysaccharide (O antigen) was obtained from P. mirabilis Bprz 86 LPS through mild acid degradation, and the six-constituent structure of its repeating unit was determined using chemical analyses and 1D and 2D ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy. It includes (R)-3-hydroxybutanoyl, which, along with fucosamine and glucose residues, forms a fragment also present in the O17 antigens. Based on the obtained serological and chemical data, the two studied P. mirabilis isolates were proposed as candidates for a new successive O serogroup in the genus Proteus, O86. Full article

Klebsiella pneumoniae is well recognized as a serious cause of infection in healthcare-associated settings and immunocompromised individuals; however, accumulating evidence from resource-limited nations documents an alarming rise in community-acquired K. pneumoniae infections, manifesting as bacteremia and pneumonia as well as neonatal sepsis. The emergence of hypervirulent and antibiotic-resistant K. pneumoniae strains threatens treatment options for clinicians. Effective vaccination strategies could represent a viable alternative that would both preempt the need for antibiotics to treat K. pneumoniae infections and reduce the burden of K. pneumoniae disease globally. There are currently no approved K. pneumoniae vaccines. We review the evidence for K. pneumoniae lipopolysaccharide (LPS) as a vaccine and immunotherapeutic target and discuss the role of antibodies specific for the core or O-antigen determinants within LPS in protection against Klebsiella spp. disease. We expand on the known role of the Klebsiella spp. capsule and O-antigen modifications in antibody surface accessibility to LPS as well as the in vitro and in vivo effector functions reported for LPS-specific antibodies. We summarize key hypotheses stemming from these studies, review the role of humoral immunity against K. pneumoniae O-antigen for protection, and identify areas requiring further research. Full article

The interaction forces and mechanical properties of the interaction between melittin (Mel) and lipopolysaccharide (LPS) are considered to be crucial driving forces for Mel when killing Gram-negative bacteria (GNB). However, how their interaction forces perform at the single-molecule level and the dissociation kinetic characteristics of the Mel/LPS complex remain poorly understood. In this study, the single-molecule-level interaction forces between Mel and LPSs from E. coli K-12, O55:B5, O111:B4, and O128:B12 were explored using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). AFM-based dynamic force spectroscopy (DFS) and an advanced analytical model were employed to investigate the kinetic characteristics of the Mel/LPS complex dissociation. The results indicated that Mel could interact with both rough (R)-form LPS (E. coli K-12) and smooth (S)-form LPSs (E. coli O55:B5, O111:B4, and O128:B12). The S-form LPS showed a more robust interaction with Mel than the R-form LPS, and a slight difference existed in the interaction forces between Mel and the diverse S-form LPS. Mel interactions with the S-form LPSs showed greater specific and non-specific interaction forces than the R-form LPS (p < 0.05), as determined by AFM-based SMFS. However, there was no significant difference in the specific and non-specific interaction forces among the three samples of S-form LPSs (p > 0.05), indicating that the variability in the O-antigen did not affect the interaction between Mel and LPSs. The DFS result showed that the Mel/S-form LPS complexes had a lower dissociation rate constant, a shorter energy barrier width, a longer bond lifetime, and a higher energy barrier height, demonstrating that Mel interacted with S-form LPS to form more stable complexes. This research enhances the existing knowledge of the interaction micromechanics and kinetic characteristics of Mel and LPS at the single-molecule level. Our research may help with the design and evaluation of new anti-GNB drugs. Full article

Salmonella enterica is a major food-borne pathogen causing food poisoning. The use of bacteriophages as alternative biocontrol agents has gained renewed interest due to the rising issue of antibiotic-resistant bacteria. We isolated and characterized three phages targeting Salmonella: SPN3US, SPN3UB, and SPN10H. Morphological and genomic analyses revealed that they belong to the class Caudoviricetes. SPN3UB, SPN3US, and SPN10H specifically target bacterial surface molecules as receptors, including O-antigens of lipopolysaccharides, flagella, and BtuB, respectively. The phages exhibited a broad host range against Salmonella strains, highlighting their potential for use in a phage cocktail. Bacterial challenge assays demonstrated significant lytic activity of the phage cocktail consisting of the three phages against S. typhimurium UK1, effectively delaying the emergence of phage-resistant bacteria. The phage cocktail effectively reduced Salmonella contamination in foods, including milk and pork and chicken meats, during cold storage. These results indicate that a phage cocktail targeting different host receptors could serve as a promising antimicrobial strategy to control Salmonella. Full article

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2–3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI–MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass. Full article

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli. Full article

In most Gram-negative bacteria, outer membrane (OM) lipopolysaccharide (LPS) molecules carry long polysaccharide chains known as the O antigens or O polysaccharides (OPS). The OPS structure varies highly from strain to strain, with more than 188 O serotypes described in E. coli. Although many bacteriophages recognize OPS as their primary receptors, these molecules can also screen OM proteins and other OM surface receptors from direct interaction with phage receptor-binding proteins (RBP). In this review, I analyze the body of evidence indicating that most of the E. coli OPS types robustly shield cells completely, preventing phage access to the OM surface. This shield not only blocks virulent phages but also restricts the acquisition of prophages. The available data suggest that OPS-mediated OM shielding is not merely one of many mechanisms of bacterial resistance to phages. Rather, it is an omnipresent factor significantly affecting the ecology, phage–host co-evolution and other related processes in E. coli and probably in many other species of Gram-negative bacteria. The phages, in turn, evolved multiple mechanisms to break through the OPS layer. These mechanisms rely on the phage RBPs recognizing the OPS or on using alternative receptors exposed above the OPS layer. The data allow one to forward the interpretation that, regardless of the type of receptors used, primary receptor recognition is always followed by the generation of a mechanical force driving the phage tail through the OPS layer. This force may be created by molecular motors of enzymatically active tail spikes or by virion structural re-arrangements at the moment of infection. Full article

The aim of the study was the serological and structural characterization of the lipopolysaccharide (LPS) O antigen from P. mirabilis Dm55 coming from the urine of a patient from Lodz. The Dm55 LPS was recognized in ELISA only by the O54 antiserum, suggesting a serological distinction of the Dm55 O antigen from all the 84 Proteus LPS serotypes described. The obtained polyclonal rabbit serum against P. mirabilis Dm55 reacted in ELISA and Western blotting with a few LPSs (including O54), but the reactions were weaker than those observed in the homologous system. The LPS of P. mirabilis Dm55 was subjected to mild acid hydrolysis, and the obtained high-molecular-mass O polysaccharide was chemically studied using sugar and methylation analyses, mass spectrometry, and ¹H and ¹³C NMR spectroscopy, including ¹H,¹H NOESY, and ¹H,¹³C HMBC experiments. The Dm55 O unit is a branched three-saccharide, and its linear fragment contains α-GalpNAc and β-Galp, whereas α-GlcpNAc occupies a terminal position. The Dm55 OPS shares a disaccharide epitope with the Proteus O54 antigen. Due to the structural differences of the studied O antigen from the other described Proteus O polysaccharides, we propose to classify the P. mirabilis Dm55 strain to a new Proteus O85 serogroup. Full article

Nosocomial outbreaks of multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) are often reported worldwide, mostly associated with a small number of multilocus-sequence types of E. hormaechei and E. cloacae strains. In Europe, the largest clonal outbreak of bla_NDM-1-producing ECC has been recently reported, involving an ST182 E. hormaechei strain in a Greek teaching hospital. In the current study, we aimed to further investigate the genetic make-up of two representative outbreak isolates. Comparative genomics of whole genome sequences (WGS) was performed, including whole genome-based taxonomic analysis and in silico prediction of virulence determinants of the bacterial cell surface, plasmids, antibiotic resistance genes and virulence factors present on genomic islands. The enterobacterial common antigen and the colanic antigen of the cell surface were identified in both isolates, being similar to the gene clusters of the E. hormaechei ATCC 49162 and E. cloacae ATCC 13047 type strains, whereas the two strains possessed different gene clusters encoding lipopolysaccharide O-antigens. Other virulence factors of the bacterial cell surface, such as flagella, fimbriae and pili, were also predicted to be encoded by gene clusters similar to those found in Enterobacter spp. and other Enterobacterales. Secretion systems and toxin–antitoxin systems, which also contribute to pathogenicity, were identified. Both isolates harboured resistance genes to multiple antimicrobial classes, including β-lactams, aminoglycosides, quinolones, chloramphenicol, trimethoprim, sulfonamides and fosfomycin; they carried bla_TEM-1, bla_OXA-1, bla_NDM-1, and one of them also carried bla_CTXM-14, bla_CTXM-15 and bla_LAP-2 plasmidic alleles. Our comprehensive analysis of the WGS assemblies revealed that bla_NDM-1-producing outbreak isolates possess components of the bacterial cell surface as well as genomic islands, harbouring resistance genes to several antimicrobial classes and various virulence factors. Differences in the plasmids carrying β-lactamase genes between the two strains have also shown diverse modes of acquisition and an ongoing evolution of these mobile elements. Full article

Background: Brucella abortus is the main causative agent for bovine brucellosis. B. abortus A19 is a widely used vaccine strain to protect cows from Brucella infection in China. However, A19 has a similar lipopolysaccharide (LPS) antigen to that of the field virulent Brucella strain, whose immunization interferes with the serodiagnosis of vaccinated and infected animals. [Aim] To develop a novel Brucella DIVA vaccine candidate. Study design and methods: The B. abortus mutant A19mut2 with the formyltransferase gene wbkC is replaced by an acetyltransferase gene wbdR from E. coli O157 using the bacterial homologous recombination technique, generating a modified O-polysaccharide that cannot induce antibodies in mice against wild-type Brucella LPS. The biological phenotypes of the A19mut2 were assessed using a growth curve analysis, agglutination tests, Western blotting, and stress resistance assays. Histopathological changes and bacterial colonization in the spleens of vaccinated mice were investigated to assess the residual virulence and protection of the A19mut2. Humoral and cellular immunity was evaluated by measuring the levels of IgG, IgG subtypes, and the release of cytokines IFN-γ and IL10 in the splenocytes of the vaccinated mice. ELISA coated with wild-type LPS can distinguish mouse antibodies induced by A19 and A19mut2 immunization. Results: The A19mut2 showed a decreased residual virulence in mice, compared to the A19 strain, but induced significant humoral and cellular immune responses, as the A19 immunization did. The protection efficacy of A19mut2 immunization against B. abortus S2308 Nal^R infection was similar to that of A19 immunization. Conclusion: The A19mut2 has potential as a novel DIVA vaccine candidate in the future. Full article

Helicobacter pylori has a unique lipopolysaccharide structure that is essential in maintaining its cell envelope integrity and imbues the bacterium with natural resistance to cationic antimicrobial peptides (CAMPs). Our group has recently elucidated the complete set of LPS glycosyltransferase genes in H. pylori reference strain G27. Here, with a series of eight systematically constructed LPS glycosyltransferase gene mutants (G27ΔHP1578, G27ΔHP1283, G27ΔHP0159, G27ΔHP0479, G27ΔHP0102, G27ΔwecA, G27ΔHP1284 and G27ΔHP1191), we investigated the roles of H. pylori LPS glycosyltransferases in maintaining cell morphology, cell wall permeability, and antimicrobial susceptibilities. We demonstrated that deletion of these LPS glycosyltransferase genes did not interfere with bacterial cell wall permeability, but resulted in significant morphological changes (coccoid, coiled “c”-shape, and irregular shapes) after 48 h growth as compared to the rod-like cell shape of the wild-type strain. Moreover, as compared with the wild-type, none of the LPS mutants had altered susceptibility against clarithromycin, levofloxacin, amoxicillin, tetracycline, and metronidazole. However, the deletion of the conserved LPS glycosyltransferases, especially the O-antigen-initiating enzyme WecA, displayed a dramatic increase in susceptibility to the CAMP polymyxin B and rifampicin. Taken together, our findings suggest that the LPS glycosyltransferases play critical roles in the maintenance of the typical spiral morphology of H. pylori, as well as resistance to CAMPs and rifampicin. The LPS glycosyltransferases could be promising targets for developing novel anti-H. pylori drugs. Full article